Profound salivary gland hypofunction and xerostomia (dry mouth) are increasingly common occurrences and are often the consequences of: 1) Sjogren's Syndrome, an autoimmune disease that targets saliva-secreting acinar tissue, 2) radiation and chemotherapeutic regimens for head and neck cancers and 3) adverse side effects from thousands of medications. Current xerostomia-based treatments are inadequate and temporary thus creating a significant clinical need for long-term solutions that include replacing irreversibly damaged or lost salivary tissue, with functional tissue grown on artificially engineered biocompatible scaffolds. However, the considerable challenge of stimulating and/or maintaining salivary epithelial cell differentiation in artificially engineered tissues has been hindered by the lack of knowledge of precise molecular signaling mechanisms that control glandular structure and function. The proposed study will address these gaps in our understanding of tissue formation by exploring the role of GTPase Rac signaling in the regulation of salivary gland branching morphogenesis and tissue polarization, processes that are crucial for development of a functional organ. The knowledge gained from these studies will further be used to investigate the role of Rac in promoting salivary epithelial cell organization and polarization on artificially engineered 3D nanoscale scaffolds, towards the future goal of generating functional artificial salivary gland constructs. The specific aims are to: (1) determine whether Rac1 GTPase is required for salivary gland branching morphogenesis and the establishment of apico-basal tissue polarity, and (2) determine whether Rac activation can promote salivary epithelial cell polarization on artificially engineered, biocompatible PLGA nanofibrous scaffolds. We will use an ex vivo whole organ culture system to examine Rac1 function in the mouse embryonic submandibular salivary gland and live time-lapse or fixed confocal microscopy to image the dynamics of branching morphogenesis and apico-basal polarity. Data will also be analyzed using biochemical immunoblotting and QRT-PCR techniques and rigorously quantified using imaging software, image segmentation and computational Cell graph methods. Salivary gland diseases like Sjvgren's syndrome, salivary adenocarcinomas and xerostomia, all feature salivary gland hypofunction as a cause, which poses an enormous burden to affected individuals, their families and the health care system as a whole. The knowledge gained from this project on the signaling mechanisms underlying early salivary gland organogenesis will be of considerable significance to the fields of tissue engineering and regenerative medicine and to future studies examining the function and possible deregulation of Rac signaling in salivary gland diseases. Abbreviations used in proposal: 2D, two-dimensional; 3D, three-dimensional; BM, basement membrane; ECM, extracellular matrix; GEF, guanine nucleotide exchange factor; GTPase, guanosine triphosphate hydroxylase; IB, immunoblotting; IF, immunofluorescence; IP, immunoprecipitation; Par, partitioning-defective proteins; PLGA, polylactic-co-glycolic acid polymer; Rac1, Ras-related C3 botulinum substrate 1; SMG, submandibular salivary gland; Tiam1, T-cell lymphoma invasion and metastasis-inducing protein 1. PUBLIC HEALTH RELEVANCE: Profound salivary gland hypofunction is a common feature in a majority of patients treated for salivary gland diseases such as Sjvgren's syndrome (SS), salivary neoplasms, adults being treated for head and neck cancer and those taking medications with anti-sialogogue sequelae. Current xerostomia-based treatments are inadequate, temporary and include pharmacological and gustatory stimulants. More long term solutions include tissue replacement and regeneration therapies, however, the significant challenge of maintaining and stimulating epithelial cell organization and differentiation in engineered tissues, remains. Since epithelial cell secretory function is crucial to organ function, understanding the cellular mechanisms regulating and maintaining tissue structure and differentiation is critical to regenerating or engineering functional tissues. The data obtained from this grant will advance basic scientific knowledge regarding novel roles for the small GTPase Rac in the control of salivary gland branching morphogenesis. Utilizing three-dimensional ex vivo whole organ culture systems, we will examine how Rac GTPase-mediated signaling pathways can control major developmental processes such as salivary gland branching morphogenesis and the formation of tissue polarity, an indispensable requirement for unidirectional and controlled flow of saliva. More significantly, the role of Rac in promoting salivary epithelial cell organization and polarization on biocompatible 3D nanofibrous scaffolds will be examined, towards the future goal of creating an artificial salivary gland construct on biocompatible scaffolds for use in regenerative medicine.